The invention relates to optimized CD4 mimic peptides derived from the CD4M33 peptide (Martin et al., Nat. Biotechnol., 2003, 21, 71-76 and International PCT Application WO 02/059146) and to the use of these peptides for the manufacture of new anti-HIV medicines and vaccines.
The human immunodeficiency virus (HIV) has been implicated as the primary cause of the slowly degenerative immune system disease termed acquired immune deficiency syndrome (AIDS). In humans, HIV replication occurs prominently in CD4 T lymphocyte populations, and HIV infection leads to depletion of this cell type and eventually to immune incompetence, opportunistic infections, neurological dysfunctions, neoplastic growth, and ultimately death.
HIV-1 treatment includes a combination of anti-HIV compounds, which target the HIV reverse transcriptase (azidothymidine (AZT), lamivudine (3TC), dideoxyinosine (ddI), tenofovir, neviparine, efavirenz), or protease (saquinavir, nelfinavir, indinavir, amprenavir, lopinavir), and only one new fusion inhibitor, enfuvirtide, has been recently approved (Richman, D. D., Nature, 2001, 410, 995-1001; Lalezari et al., N. Engl. J. Med., 2003, 348, 2175-2185). However, the emergence of new HIV isolates resistant to existing drugs, in addition to difficulties in compliance with drug regimens because of pill burden and adverse side effects, suggests that new therapies with new drugs targeting different steps of the HIV cycle are urgently needed.
Although considerable effort has been expended on the design of effective vaccine, currently no vaccine against HIV infection exists.
The HIV viral particle comprises a viral core composed of capsid proteins, RNA genome and enzymes, surrounded by a shell of myristylated gag proteins. This shell is in turn surrounded by an outer lipid membrane envelope comprising the HIV envelope glycoproteins (gp120 and gp41). The HIV envelope glycoproteins are synthesized as a single 160 kilodalton precursor protein, which is cleaved by a cellular protease during viral budding into two glycoproteins, gp41 and gp120. gp41 is a transmembrane glycoprotein and gp120 is an extracellular glyco-protein, which remains non-covalently associated with gp41. gp120 is displayed as a gp41-associated trimer and forms envelope spikes on the surface of HIV virions.
The HIV entry is a multiple-step process initiated by the binding of the HIV surface envelope glycoprotein gp120 (Env) to the host cell CD4 receptor. This association induces conformational changes in Env that allow its binding to a chemokine co-receptor CCR5 or CXCR4 (Wu et al., Nature, 1996, 384, 179-183; Trkola et al., Nature, 1996, 384, 184-187; Feng et al., Science, 1996, 272, 872-877). Association with this co-receptor activates the fusogenic properties of the non-covalently associated gp41 transmembrane protein and subsequent entry of the virus into the cell (Wyatt R. and Sodroski J., Science 1998, 280, 1884-1888).
Each of these steps can represent a potential target for new drugs (Blair et al., Drug Discov. Today, 2000, 5, 183-194; Moore J. P. and Doms R. W., P.N.A.S., 2003, 100, 10598-10602; Vermeire, K., and Schols, D., Expert. Opin. Investig. Drugs, 2005, 14, 1199-1212; Ryser H. J. P and Flückiger, R., Drug discovery today, 2005, 10, 1085-1094).
Information on the cellular receptors involved in virus infection, as well as on the viral envelope structure and its interaction with host cells, may help in the design of entry inhibitors and HIV vaccines.
The three-dimensional structure of the gp120 “core” protein has been determined in the CD4-bound conformation (gp120HXB2:CD4:17b complex; PDB code 1g9m; Kwong et al., Nature, 1998, 393, 648-659; Huang et al., Science, 2005, 310, 1025-1028; Kwong et al., Structure, 2000, 8, 1329-1339) and the more recently published unliganded form of SIV gp120 (PDB code 2BF1; Chen et al., Structure, 2005, 13, 197-211), but so far there is no crystal structure available of the gp120 trimer.
In the CD4-bound conformation, gp120 consists in an inner and an outer domain connected by a four-stranded β-sheet (bridging sheet), whereas in the unliganded conformation, although it maintains this two-domain organization, the inner domain is significantly different and the β-sheet is not formed. CD4 binding creates a cavity of roughly 150 Å3, which extends deeply in the interior of gp120 at the intersection between the inner and outer domain, whereas this cavity is absent in the unliganded form.
In the complex, a large surface of the domain D1 (742 Å2) of CD4 binds to a large (800 Å2) conserved depression on gp120. The CD4 interface is comprised of 12 residues (positions 36 to 47 of CD4 amino acid sequence corresponding to the CDR2-like loop of CD4) contributing to gp120 binding with mixed hydrophobic, electrostatic, H-bonding interactions. In the complex, CD4 Phe43 side chain plugs the entrance of the gp120 cavity (Phe43 cavity or Phe43 pocket) and CD4 Arg59, just behind Phe43, is involved in a double H-bond with Asp368 in gp120.
Besides these cell receptors, HIV is capable of binding to other molecules present on the cells that it infects, such as DC-SIGN, sphingolipides or heparan sulphates. Heparin, sulfated polysaccharides and polyanions in general are known to bind to the V3 loop of the viral envelope gp120 (with a preference for envelopes of X4 tropism), (Harrop, H. A. and Rider, C. C., Glycobiol., 8, 131-137; Moulard et al, J. Virol., 2000, 74, 1948-1960) and to a CD4-induced (CD4i) region of gp120, close to V3, involved in co-receptor binding (Vivès et al., J. Biol. Chem., 2005, 280, 21353-21357). The association between V3 loop and those molecules seems to dominate the electrostatic effect of this double interaction and probably occurs through interactions between the acidic sulfate moieties of heparin derivatives and basic residues of V3 loop. Viruses of X4 tropism are known to have more basic V3 loops (Berger et al., Nature, 1998, 391, 240-) and would therefore be better binders to heparin derivatives. This does not exclude an affinity of heparin derivatives to the CD4i epitope of viruses of R5 tropism, since peptides containing sulfated tyrosine are also able to associate with those gp120 (Farzan et al., J. Biol. Chem., 2002, 277, 40397-40402).
Cell attachment is the first step in HIV-1 entry and a primary target for antiviral therapy and vaccine design.
Antiviral-Therapy
Different macromolecules have been demonstrated to inhibit gp120 binding to CD4, starting from soluble CD4 (Daar et al., P.N.A.S., 1990, 87, 6574-6578). However, monovalent potent inhibitors of CD4-gp120 binding such as soluble CD4 are shown to be effective in vitro (Daar et al., precited) but have reduced affinity for primary isolates. Evidence is arising that HIV-1 through its envelope trimers could bind several cell-surface CD4 receptors simultaneously (Kwong et al., Nature, 2002, 420, 678-682). Multimeric inhibitors targeting several CD4 binding sites on single spike or even on a virion could therefore be much more able to compete with CD4 for the attachment to the virus. Well-tailored multivalent ligands could lead to large avidity gains by decreasing the off-rate of the complex and increasing functional affinity of the ligand (Gestwicki et al., J. Am. Chem. Soc., 2002, 124, 14922-14933; Sadler et al., Rev. Mol., Biotech., 2002, 90, 195-229).
Only few multimeric compounds displaying several molecules of CD4 have been developed so far. Among them, complex constructs presenting four or twelve copies of CD4 domains in an immunoglobulin structure were reported (Allaway et al., AIDS Res. Hum. Retroviruses, 1995, 11, 533-539; Gauduin et al., J. Virol., 1996, 70, 2586-2592; Arthos et al., J. Biol. Chem., 2002, 277, 11456-11464) and led to promising results as HIV-1 inhibitors (Arthos et al., precited; Trkola et al., J. Virol., 1995, 69, 6609-6617). The increased stability of these molecules and the possibility that they may simultaneously block several gp120 subunits of the trimeric envelope at the surface of virions or spikes of infected cells may explain their superior antiviral potency. Nevertheless, the large size of these molecules and the possibility that they may induce an anti-CD4 auto-immune response might represent a limitation for their therapeutic applications.
However, in spite of many years of efforts worldwide, only a handful of small molecules targeting CD4 binding site on gp120 and inhibiting CD4 attachment has been discovered. The large size and complexity of the CD4 interface make the reproduction of such functional epitope into a small molecule a challenge, and explain the difficulty in the development of small molecule inhibitors of gp120-CD4 interaction.
For some time, the recently-described small molecule BMS-378806 developed by Bristol-Myers Squibb (Wang et al., J. Med. Chem., 2003, 46, 4236-4239) was believed to inhibit CD4-gp120 binding but more recent studies have demonstrated that BMS would interact with another region of gp120, thus hindering the conformational changes induced by CD4 binding (Si et al., P.N.A.S., 2004, 101, 5036-5041; Madani et al., J. Virol., 2004, 78, 3742-3752). This molecule was dimerized with a low increase in activity (Wang et al., Org. Biomol. Chem., 2005, 3, 1781-1786).
The International PCT Application WO 2005/121175 describes small molecule CD4 mimetics comprising fused bicyclic or tricyclic core structure. However, no antiviral activity has been demonstrated for these molecules.
Until now, the CD4 mimics designed from scorpion toxin scyllatoxin have remained the smallest potent inhibitors of that kind available (Vita et al., P.N.A.S., 1999, 96, 13091-13096; Martin et al., Nature Biotech., 2003, 21, 71-76; Stricher et al., Biochem. J., 2005, 390, 29-39; International PCT Application WO 02/059146). The mini-proteins mimic of CD4 were designed to reproduce the structure of the gp120 glycoprotein binding “hot spot” of the CD4 surface, on to the scaffold consisting of the scorpion (Leiurus quinquestriatus hebraeus) toxin scyllatoxin. This small (31-residues) toxin was selected since its structure, formed by an antiparallel β-sheet linked to a short helix by three disulphide bridges, contains an exposed positions 18-29 β-hairpin, which could superimpose its backbone atoms on those of positions 36-47 CDR2-like loop of CD4 with an r.m.s (root mean square) deviation of only 1.10 Å. On the basis of this structural similarity, the scaffold permissiveness in sequence mutations and stability, even after sequence replacements, critical functional residues of the CDR2-like loop of CD4 could be grafted on to the β-hairpin of scyllatoxin, leading to the initial low-affinity mimic (CD4M3).
A first optimization of the interactions with gp120 was achieved by structure-activity studies, leading to the first generation of mini-proteins, CD4M9. CD4M9 is able to inhibit the binding of soluble CD4 to gp120 with an IC50 (concentration causing 50% inhibition of sCD4 binding to gp120) about a hundred-fold higher than that of native CD4. Recently Li et al. have reported a dimeric version of the first generation of CD4 mimic M9 with a twenty-fold enhanced anti-HIV activity over the monovalent molecule (Li et al., Bioconj. Chem., 2004, 15, 783-789 and US Patent Application 2005/0176642).
Further optimization was achieved with the help of the NMR structure of the CD4M9 mini-protein combined to molecular modeling, leading to the second generation of mini-proteins, CD4M33 (27 amino acids, SEQ ID NO: 1). CD4M33 is able to bind different gp120s in competition with soluble CD4 with a nanomolar Kd, to induce CD4-like conformational changes in gp120, exposing the cryptic epitopes necessary to target co-receptor elements, as well as to inhibit infection of primary cells by primary clinical HIV-1 isolates.
In addition, heparin or heparin fragments of sufficient size, in the presence of CD4 mini-protein interact with the CD4i domain of the gp120 and this combination greatly inhibits the gp120/co-receptors interaction, as demonstrated by inhibition of the gp120/48D or 17b antibody interaction (International PCT Application WO 03/089000).
However, HIV-1 inhibition in cell-cell fusion and virus-cell fusion assays remains less efficient with these mimics than that with CD4s.
Some of those small and stable mini-proteins were co-crystallized in complex with gp120 and antibody 17b Fab fragment (Huang et al., Structure 2005, 13, 755-768), providing precise structural information about the binding of those compounds with the gp120 binding site. The three-dimensional structure of CD4M33, free or in complex with gp120 (PDB code 1YYL) has been solved (Stricher et al., Biochem. J., 2005, 390, 29-39; Huang et al., precited). In CD4M33, Biphenylalanine 23 was shown to play a key role in the interaction with gp120 binding pocket. Another important structural feature of this mini-protein is the β-hairpin which represents the “hotspot” of the binding region to gp120 (FIG. 1), accounting with about 80% of the interface. However, the residues defining this β-turn have not been well explored in the previous studies.
HIV-Vaccine
gp120 appears to be the primary target for inducing a humoral immune response to HIV. However, it has been difficult to generate protective responses against the HIV Env because the CD4 binding site is buried between the outer domain, the inner domain, and the V1/V2 domains of gp120. Thus, although deletion of the V1/V2 domain may render the virus more susceptible to neutralization by monoclonal antibody directed to the CD4 site, the conformation of Env prior to CD4 binding may prevent an antibody response.
It has been shown that CD4 and CD4 mimetics that bind to gp120 cause a conformational change in Env that exposes one or more cryptic or inducible epitopes in or near the CD4 binding site, which in turn allows the generation of a neutralizing antibody response to Env.
Therefore, the use of complexes of Env and CD4 or Env and CD4 mimics (mini-proteins derived from scyllatoxin or small cyclic molecules) as vaccine to generate a protective immune response against HIV has been proposed (International PCT Applications WO 2004/037847 and WO 2005/121175).
The use of small CD4 mimics should prevent the risk of inducing an anti-CD4 auto-immune response that might occur when using the CD4 molecule. Unlike CD4, CD4M33 was shown to be low immunogenic as indicated by lower level and antibodies induced by the mini-protein. Moreover, anti-CD4M33 antibodies did not cross-react with CD4. Therefore, the use of CD4M33 in a vaccine may be safer compared to CD4.